Investigating the Role of XBP1 Unconventional Intron Splicing in the Unfolded Protein Response

The primary and general objective of this project was to investigate whether a 26-nucleotide unconventional intron of X-box binding protein 1 (XBP1) that is being spliced out by inositol-requiring enzyme 1α (IRE1α), plays a role in the unfolded protein response (UPR). XBP1 is a transcription factor that undergoes unconventional splicing, catalyzed by inositol-requiring enzyme 1α (IRE1α), in response to endoplasmic reticulum stress.

   Experimental approach

The initial task, crucial for the subsequent experimental steps, was the design and optimization of a real-time PCR method for detecting and quantifying the XBP1 intron. To that end, we adapted the approach by Tong et al. [1] based on SYBR Green dye fluorescence which involves a two-step reaction:

1)      Reverse transcription (RT) of the XBP1 intron using a specific RT primer.

2)      Real-time PCR reaction with SYBR Green- based detection.

We designed a set of three primers:

-        An RT primer containing a stem-loop structure and 3’-terminal 11-nucleotide fragment complementary to XBP1 intron.

-        A forward (fwd) primer containing a 12-nucleotide fragment complementary to XBP1 intron.

-        A reverse (rev) primer complementary to the stem-loop region of the RT primer.

The length of PCR product (68 bp) was within the recommended range (50-210 bp), making it sufficiently long and suitable for detection by SYBR Green fluorescence.

The primer sequences and an outline of the approach used (Fig. 1):

RT primer:  CGTCTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACGCCAGAGGTGCAC

intron fwd primer:      TTGAGCACCAGCACTCAGAC

intron rev primer:      TGGTGTCGTGGAGTCGGC

Fig. 1. Outline of the experimental approach

Additionally, two sets of PCR primers were designed:

-        Primers for detecting the spliced version of the XBP1 transcript (XBP1s), adapted from Yoon et al. [2], with a PCR product length of 130 bp:

XBP1s fwd primer: GCTGAGTCCGCAGCAGGT

XBP1s rev primer: CTGGGTCCAAGTTGTCCAGAAT

-        Primers for detecting the superoxide dismutase 1 (SOD1) transcript, used as a reference gene, with a PCR product length of 129 bp:

SOD1 fwd primer:  CTCACTCTCAGGAGACCATTGC

SOD1 rev primer:  CCACAAGCCAAACGACTTCCAG

The specificity of both primer pairs was confirmed by Primer-BLAST check.

The RT reactions were performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The specific RT primer was used for the XBP1 intron RT reaction, while RT random primers were used for the XBP1s and SOD1 RT reactions. The resulting RT reaction mixes were then used as templates for real-time PCR with the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and the specific PCR primers.

eExperiments and results

HeLa S3 cells were seeded onto 6-well plates, and ER stress was induced using tunicamycin (Tm) at a concentration of 2.5 µg/ml. Additionally, cells were treated with the specific IRE1α inhibitor, 4µ8C, at a concentration of 22.5 µM. Samples were collected after 4 and 8 hours of exposure to Tm, and total RNA was isolated using the RNeasy Kit (Qiagen). The RNA samples were then used as templates for RT and PCR reactions.

Real-time PCR reactions for XBP1s and SOD1 yielded technically accurate results (Fig. 2). As expected, Tm induced the UPR response and XBP1 splicing, which was inhibited by the presence of 4µ8C (Fig. 3). Unfortunately, we were not able to detect the XBP1 intron in real-time PCR. Attempts to optimize primer concentrations (50/100 nM) and annealing temperature (from 50°C to 65°C) were unsuccessful (Fig. 4).

Fig. 2. Amplification plots for XBP1s (A) and SOD1 (B) and melt curves for XBP1s (C) and SOD1 (D).

Fig. 3. XBP1s relative expression levels (SOD1 used as a reference gene) in HeLa cells exposed to Tm and 4µ8C.

Fig. 4. Representative amplification plots (A) and melt curves (B) for XBP1s intron (annealing temperature 56°C).

  Conclusions

Despite our efforts, we were unable to achieve successful amplification of the XBP1 intron in the real-time PCR reaction. The reasons for this may be either technical, such as issues with primer design, or biological, as the XBP1 intron may be quickly degraded in the cell after excision by IRE1α, making it undetectable by PCR.

References:

[1] Tong L. et al. Improved RT-PCR Assay to Quantitate the Pri-, Pre-, and Mature microRNAs with Higher Efficiency and Accuracy. Mol. Biotechnol. 2015; 57(10):939-46. DOI: 10.1007/s12033-015-9885-y

[2] Yoon SB. et al. Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method. PLoS One 2019; 14(7):e0219978. DOI: 10.1371/journal.pone.0219978